Tracking DOT1L methyltransferase activity by stable isotope labelling using a selective synthetic co-factor

Epigenetic processes influence health and disease through mechanisms which alter gene expression. In contrast to genetic changes which affect DNA sequences, epigenetic marks include DNA base modifications or post-translational modification (PTM) of proteins. Histone methylation is a prominent and versatile example of an epigenetic marker: gene expression or silencing is dependent on the location and extent of the methylation. Protein methyltransferases exhibit functional redundancy and broad preferences for multiple histone residues, which presents a challenge for the study of their individual activities. We developed an isotopically labelled analogue of co-factor S-adenosyl-L-methionine (13CD3-BrSAM), with selectivity for the histone lysine methyltransferase DOT1L, permitting tracking of methylation activity by mass spectrometry (MS). This concept could be applied to other methyltransferases, linking PTM discovery to enzymatic mediators.


In vitro methylation reactions
The methyltransferase enzymes SETD7, EHMT1, SETD2, SUB420H2 were kindly donated by Prof. M. Vedadi (University of Toronto).The catalytically competent recombinant DOT1L truncated protein was purchased from Life Technologies Ltd (11552HNCE250) and HeLa derived nucleosomes from AMSBIO (52039).Reactions conditions were performed as described here 1 (see also Supplementary Table 2).

Cell Lysate Methylation Reactions
HEK293T cells were cultured in T75 flasks with DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere at 37 °C with 5% CO2.Cells were maintained at 50-80% confluency and >95% viability.Prior to cell lysis, cells were treated with 0.1 µM of the DOT1L inhibitor EPZ5676 (Stratech, S7062) for 4 days.Lysates were prepared by washing cells 3x with chilled PBS and recovered from the surface of a cell culture flask with 1 mL Trypsin/EDTA solution.Cells were centrifuged for 5 minutes at 300 xg at 4 °C to remove the trypsin and then suspended in 0.1 mL 50 mM Tris-HCl, 5 mM MgCl2, 4 mM DTT, 0.1% NP-40, 0.2 mM PMSF and 10 units/mL DNase and left on ice for 5 minutes.After douncing cells (60 strokes on ice), the lysate was centrifuged at 18000 xg for 10 minutes (4 °C) to remove debris and the quantity of recovered proteins was determined using a Protein Assay 2 kit (Biorad) as per the manufacturer's instructions.Reactions with DOT1L were performed for 3 hours at 37 °C in 500 µL 50 mM Tris, 5 mM MgCl2 and 4 mM DTT, as described in Supplementary Table 3.Reactions were quenched by adding 4x the sample volume (2 mL) of ice-cold methanol and immediately performing chloroform-methanol precipitation.Precipitates were dissolved in 50 µL 8 M urea in 100 mM ammonium bicarbonate and reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 minutes at 37 °C.After alkylating with 15 mM iodoacetamide (30 minutes, room temperature in the dark), the total volume was increased to 350 µL with 100 mM ammonium bicarbonate and the proteins were digested with sequencing grade modified trypsin/LysC (Promega) at 1:50 (protease:protein) overnight.Digested lysates were desalted and fractionated into 8 using Pierce high pH fractionation kit as per the manufacturer's instructions and fully dried at room temperature in a vacuum centrifuge.

LC-MS
Each sample was dissolved in 20 µL 0.1% formic acid (FA) by titration followed by sonicating for 10 minutes.After centrifuging for 2 minutes at 13,000 x g, samples were transferred to glass sample vials.5 µL was injected onto a 2 cm x 75 µm Acclaim Pepmap 100 trap column for 3 minutes, before being separated on an 15 cmx 75 µm Acclaim Pepmap RSLC column over 140 minutes, starting in 92% (5% dimethylsulfoxide (DMSO), 0.1% FA), ramping to 45% (5% DMSO, 0.1% FA, 75% acetonitrile) over 110 minutes using a Ultimate 3000 HPLC (Thermo Scientific) interfaced with an IT-FT mass spectrometry (Orbitrap Velos, Thermo Scientific) via a nano-ESI source.Mass spectrometry was performed using Top 10 CID, over a range of 200-2000 mz at 60k resolution.Singly charged parent ions were excluded from MSMS acquisition and a 20 second exclusion list (with repeat measurements within 20 seconds enabled) was enabled.Fragmentation was performed by CID at a normalized energy of 30.

Database Searching
Database searching was conducted in proteome discoverer 2.2 (Thermo Scientific) using the Mascot search engine (Matric Science), searching the Swissprot human reference proteome (Uniprot, downloaded April 2018).Carbamidomethylation was set as a fixed modification with oxidized methionine, and heavy/light methylation (mono, di, tri)-K and (mono, di)-R were set as variable modifications.Percolator was used to rescore peptides with FDR filtering set at 1% (strict) and 5% (relaxed).

MS2 Matching
MS2 spectra that gave rise to a peptide spectral match were searched against MS2 spectra arising from parent ions with a mass shift corresponding to heavy/light labeling (i.e.~4 Da shift) using an inhouse script.Briefly, mass spectrometry files were converted to mzXML using default setting in MSConvert 2 and read into R using the mzR package.For each MS2 with a PSM (from Mascot database searching), peak lists were extracted for MS2 scans arising from parent ions with a 4Da shift.Peak lists were exported and compared for matching and complementary ions as described for HiLight-PTM 3 , using a tolerance of 0.5 and 0.8 mz for matching the same and complementary-mass shift ions respectively.Pearson's product moment correlation coefficient was then calculated in R for the intensity of the matched and complementary mass-shifted ions.

Quantitative kinetic analysis of H3K79 methylation by DOT1L with 13 CD3 co-factor analogues
The K79 peptide standard used for the following quantitative kinetic analyses (Supplementary Figure 2 and 3) was purchased from the AQUA Peptide laboratory of Sigma Aldrich.The procedures for the preparation of histone peptides for MS analysis were previously published by Lin and Garcia 4 .
Reactions were carried out at 37 °C in KMT buffer (7.5  For the time course study, at the required interval (15, 30, 45 or 60 minutes) reactions were performed using 100 μM 13 CD3SAM or 13 CD3BrSAM.For the titration study, reactions were performed for 15 minutes, using concentrations between 100 nM and 1 mM of 13 CD3SAM or 13 CD3BrSAM.Reactions were quenched with the addition of 1 mM cold SAH (dissolved in 99% H2O, 1% FA).After quenching, samples were subsequently dried using a speedvac and then subjected to preparation for MS analysis.
Three replicates per data point were obtained and absolute quantitative analysis was carried out using the addition of 2 pmol of a K79un-containing peptide standard bearing a heavy labeled arginine residue at the C-terminus.The peptide standard was calibrated by addition to unreacted nucleosomes prepared for MS in a manner analogous to those obtained from reaction conditions (see Supplementary Figure 4-5).Only the unmodified and mono-methyl (light and heavy) states were investigated; the error associated with excluding heavy di-methyl signals was considered to be negligible as the total proportion of di-methylated states in a sample reaches no larger than 2.6% ( 13 CD3SAM) and 1% ( 13 CD3BrSAM) across the 60 minute time frame.
The following criteria were applied to assess MS sample quality: • Signal intensity of the peak of interest (LC) should be greater than 1 x 10  6).Peaks that match exactly will not carry the di-methyl label, whereas their counter-fragment will.Therefore, if we observe exact matched peaks between the two spectra, we can predict the presence of the labelled-equivalent, these are indicated as "Prediction and matched"."Mass-shift only" means there was no unlabeled counter-fragment identified.The intensity of the matches ions were normalised by the most intense peak in their respective spectra (base-peak normalised) and correlate with r=0.8 (P<0.001,Pearson's correlation coefficient) Supplementary Figure 5: MS1 scans showing the putative presence of H3K79 mono-methyl and dimethyl peaks.The positive control, and samples A-D, show the incorporation of heavy labelling, whereby peaks within the regions labelled 13 CD3 (light-green), 13 CD3.CH3 (purple) and 2[ 13 CD3] (pink), indicating a reaction with BrSAM-13 CD3.In the negative control (no DOT1L), only endogenous methylation (no heavy label) can be seen in the regions labelled CH3 (dark green) and 2[CH3] (orange).
Supplementary Figure 6 (a) K60 di-methylation (2[CH3]) of H2BWT was identified following database searching, and (b) an MS2 spectra from a precursor corresponding to a +4 shift was identified from (c) an in-house script that looks for peaks that are common (green) or complementary (orange with blue dot/mass annotation).

Chemical synthesis
General methods 7-bromo-6-chloro-7-deazapurine 1 was purchased from Fluorochem, all other chemicals were purchased from Sigma Aldrich.SAH, SAM and related compounds were stored below -20 o C. All solvents used were dry unless otherwise indicated.Reactions were performed on an IKA magnetic hotplate stirrer unless otherwise stated.Microwave irradiation was carried out using a Discoverer SP system (CEM Technology).Reactions were monitored using TLC plates purchased from Merck (60 F254, 0.25 mm).Column chromatography was carried out using either silica gel (40-63 μm 60 Å, Merck) for normal phase separation or Amberlite XAD-4 resin (Sigma Aldrich) for reverse phase separation.Isolated compounds were dried using a Büchi R-114 rotary evaporator.HPLC fractions were dried using a speedvac concentrator (Thermo Scientific Savant).Compound purity was assessed by TLC, NMR and MS. 1 H and 13 C NMR for the majority of compounds were recorded using a Bruker Advance 400 Spectrometer.The samples were prepared in an appropriate deuterated solvent (purchased from Cambridge Isotope Laboratories or Sigma Aldrich) and the internal reference was Me4Si.For compound 7, 200 µL of aqueous solution of 13 CD3-BrSAM were mixed with 20 µL of potassium phosphate buffer (1.5 M in D2O at pH 7.4, containing NaN3 2 mM and 5.8 mM TSP as a reference).The solution was introduced in a 3 mm NMR tube and analysed in a Bruker Avance III 600 equipped with a BBI probe working at room temperature.Mass spectra (electrospray ionisation or chemical ionisation: ESI, CI) were recorded by the Mass Spectrometry Service of Imperial College London, Department of Chemistry using a Micromass Autospec Premier and Micromass LCT Premier spectrometer.Alternatively, LC-MS data was obtained using an LTQ Velos Pro linear ion trap LC-MS system (Thermo Scientific).Electrospray ionisation (ESI) was the primary ionisation method over a mass range of 200−2000 m/z and a secondary CID fragmentation was applied with a collision energy of 35%.Identification of the ion of interest was conducted through product fragment comparisons with theoretical fragmentation of the desired chemical structure.The samples were eluted over a linear gradient of 100% solvent A (99.9% H2O, 0.1% FA) to 5% A and 95% B (95% MeCN, 4.9% H2O, 0.1% FA) over 10 minutes, at which point the gradient remained isocratic for 2 minutes.For HPLC purifications, crude products were fractioned by UV peak detection (215 nm) using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific) using a Supelco Ascentis Express C18 column (2.7 µm particle size, 150 mm x 4.6 mm, Sigma Aldrich).The LC conditions comprised of a flow rate of 0.5 mL/min and a multi-step gradient starting at 0.5% B (95% MeCN, 4.8% H2O and 0.2% TFA or 0.2% FA) and 99.5% A (5% MeCN, 94.8% H2O and 0.2% TFA or 0.2% FA) for two minutes, then linearly increased to 20% B over 15 minutes, followed by linear increase to 95% B over a further 7 minutes.All solvents used were HPLC grade (Sigma Aldrich or Thermo Fisher).To identify the purified peak of interest, fractions were further analyzed using LC-MS as described above.
The solvent was then removed in vacuo and replaced with 1 mL of water.The solution was cooled in an ice bath and adjusted to pH 7-8 using dropwise addition of 2 M HCl.An off-white precipitate formed and was left to stand for fifteen minutes.The supernatant liquid was removed using a syringe.This washing method was repeated three times.5 (4 mg, 0.0086 mmol) was dissolved in neat HCOOH (1 mL).A separate suspension of AgOTf (104 mg, 0.41 mmol) and CD3I (252 µL, 4.1 mmol) in CCl4 (1 mL) was stirred for 10 minutes at RT under N2, after which time a bright yellow precipitate had formed.The mixture was filtered over a syringe pad and the filtrate was added to the solution of 5. High-speed stirring was maintained for 24 hours.Water (1 mL) was added to the reaction mixture, which was subsequently dried in vacuo.The product was purified by HPLC under the conditions outlined in the general methods (the product eluted at 5.6 min)to give a clear residue (1.2 mg, 0.0020 mmol).8 was prepared from 6 (SAH) (10 mg, 0.026 mmol) and 13 CD3I using the procedure for compound 7.

Table 1 :
Summary of previous studies of protein methyltransferase (PMT) activities using the bump-and-hole approach.

Table 2 : Summary of the conditions used for nucleosome labelling experiments.
All reactions were performed at 37 °C for one hour with a total reaction volume of 0.2 mL.
Supplementary Table3: Reagent quantities for DOT1L reactions in cell lysate.Percentages in sample names relate to the proportion of 13 CD3-BrSAM as the methyl donor.

Table 4 : The observed (expt_mz) and corresponding theoretical Mz (theory_mz) for H3K79 with di-methyl heavy labelling (2[ 13 CD3] -H2)
(see Figure3cin the main manuscript).The difference in Da (dDa) and PPM (dPPM) is provided.Fragmentation data was acquired in a linear ion trap, and database-searched using Mascot with 0.6Da tolerance for fragments and 10ppm for parent ion mass.The table was generated directly from the mass spectrometry Raw files using an in-house script in R. The fragment-type is given (ms2Type) within [ ] with the theoretical charge after the brackets; a=a-ion series, b=b-ion series, p=parent ion, y=y-ion series.Intensity values are normalised by the most intense ion (base-peak normalization).

Table 5 : The observed (expt_mz) and corresponding theoretical Mz (theory_mz) for H2BWT K60 dimethyl
(2[CH2]) (see Supplementary Figure6).The difference in Da (dDa) and PPM (dPPM) is provided.Fragmentation data was acquired in a linear ion trap, and database-searched using Mascot with 0.6Da tolerance for fragments and 10ppm for parent ion mass.The table was generated directly from the mass spectrometry Raw files using an in-house script in R. The fragment-type is given (ms2Type) within [ ] with the theoretical charge after the brackets; a=a-ion series, b=b-ion series, p=parent ion, n=parent ion -NH2; w=parent ion -H2O, w= and y=y-ion series.Intensity values are normalised by the most intense ion (base-peak normalization).